Protein phosphorylation catalyzed by protein kinases is one of the most common modes of regulation of protein function. By adding phosphate groups to substrate proteins, protein kinases alter the activity, localization and overall function of many proteins and influence almost all cellular processes. At least 30% of the human proteome is estimated to be phosphorylated by protein kinases. Protein phosphorylation is particularly prominent in signal transduction. Protein kinases are implicated in a variety of diseases including inflammation, cancer, neurodegenerative disorders, diabetes, infectious diseases, and so on. The human genome is estimated to encode 518 protein kinases. Based on the residue they phosphorylate, protein kinases are classified into 2 major groups: 1) protein tyrosine kinases or PTKs (˜90 members) and 2) protein serine/threonine kinases (˜378 members). The rest are ‘atypical’ kinases. The kinase domain of all typical protein kinases is highly conserved and consists of two lobes (N-lobe and C-lobe) that surround the nucleotide binding site.
Among the PTKs, a small subfamily known as Janus family kinases (JAKs) consists of four members namely JAK1, JAK2, JAK3, and Tyk2. They are cytoplasmic protein tyrosine kinases that play essential and specific roles in immune cell development and function by participating in the cytokine receptor signal transduction. Binding of cytokines activates the JAKs which in turn phosphorylate and activate a set of transcription factors known as STAT (signal transducers and activators of transcription) proteins. The STAT proteins form homo- or heterodimers and translocate to the nucleus where they induce transcription of genes. The central role of the JAK/STAT pathways in relaying the signals from many cytokine receptors, and the involvement of several cytokines in a range of pathologies such as diseases of the immune system and cancer, makes them attractive targets for drug discovery.
Among the JAKs, JAK3 has particularly selective functions. Unlike the other members of the JAK family, which show wide tissue distribution, JAK3 expression is restricted to the cells of hematopoietic lineage. Unlike the other members of the JAK family which associate with multiple cytokine receptors, JAK3 associates uniquely with γc-chain, the common signaling subunit of receptor complexes for six cytokines namely interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These ILs play a pivotal role in the lymphoid development and function. JAK3 is inducible in T and B cells and expressed at high levels in NK cells and normally in thymocytes, platelets, mast cells. JAK3, through its association with the IL-2 receptor, is critical for lymphocyte survival, differentiation, and function. In humans, mutations in either JAK3 or γc-chain are associated with rare and inherited disorder known as severe combined immunodeficiency (SCID) indicating their critical role in the development and function of lymphocytes. These patients do not have deficits outside the immune system and hematopoietic stem cell transplants are curative, suggesting very discrete functions for JAK3.
The SCID phenotype was also observed in JAK3 knockout mice. JAK3 deficiency in humans results in the lack of T cells and NK cell development; B cells are present but their function is not normal. Unlike humans, JAK3 knockout mice show the lack of B cells and have relatively small numbers of T cells. The reason for this difference in the role of JAK3 in B cell development between mice and humans is not clear but it could be due to species-specific cytokine usage. However, similar to humans, JAK3 knockout mice did not display any effect on the development of myeloid or erythroid cells confirming the restriction of JAK3 function to lymphocyte development.
Though initially it was believed that the primary function of JAK3 is regulation of function of T and B cells through cytokine dependent pathway, recent studies using JAK3 knockout mice and JAK3 specific inhibitors suggest that JAK3 can transduce signals in non-cytokine-dependent manner in mast cells and that JAK3 plays a key role in mast cell mediated inflammatory responses. The enzymatic activity of JAK3 is increased by IgE receptor cross-linking in mast cells.
Other JAK family members Tyk2, JAK1 and JAK2 have functions within and outside immune cells. Mutations of Tyk2 cause autosomal recessive hyperIgE syndrome and JAK2 gain-of-function mutations (V617F) underlie a subset of disorders collectively referred to as myeloproliferative diseases. In some contexts, both JAK1 and JAK3 play dual and equal roles in receptor phosphorylation events indicating potential synergistic effects due to suppressing both JAK3 and JAK1 signaling.
JAK family members have been implicated in additional conditions including myeloproliferative disorders (O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506), where mutations in JAK2 have been identified. This indicates that inhibitors of JAK in particular JAK2 may also be of use in the treatment of myeloproliferative disorders. Additionally, the JAK family, in particular JAK1, JAK2 and JAK3, has been linked to cancers, in particular leukaemias e.g. acute myeloid leukaemia (O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506; Xiang et al, 2008, “Identification of somatic JAK1 mutations in patients with acute myeloid leukemia” Blood First Edition Paper, prepublished online Dec. 26, 2007; DOI 10.1 182/blood-2007-05-090308) and acute lymphoblastic leukaemia (Mullighan et al, 2009) or solid tumours e.g. uterine leiomyosarcoma (Constantinescu et al, 2007, Trends in Biochemical Sciences 33(3): 122-131), prostate cancer (Tarn et al, 2007, British Journal of Cancer, 97, 378-383). These results indicate that inhibitors of JAK, in particular of JAK1 and/or JAK2, may also have utility in the treatment of cancers (leukaemias and solid tumours e.g. uterine leiomyosarcoma, prostate cancer).
JAK1 is a novel target in the immuno-inflammatory disease area. JAK1 heterodimerizes with the other JAKs to transduce cytokine-driven pro-inflammatory signaling. Therefore, inhibition of JAK1 and/or other JAKs is expected to be of therapeutic benefit for a range of inflammatory conditions as well as for other diseases driven by JAK-mediated signal transduction.
Vandeghinste et al. (WO 2005/124342) discovered JAK1 as a target whose inhibition might have therapeutic relevance for several diseases including OA. Knockout of the JAK1 gene in mice demonstrated that JAK1 plays essential and non-redundant roles during development: JAK1−/− mice died within 24h after birth and lymphocyte development was severely impaired. Moreover, JAK1−/− cells were not, or less, reactive to cytokines that use class II cytokine receptors, cytokine receptors that use the gamma-c subunit for signaling and the family of cytokine receptors that use the gp130 subunit for signaling (Rodig et al, 1998).
Various groups have implicated JAK-STAT signaling in chondrocyte biology. Li et al JAK1 was initially identified in a screen for novel kinases (Wilks A. F., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1603-1607). Genetic and biochemical studies have shown that JAK1 is functionally and physically associated with the type I interferon (e.g., IFNalpha), type II interferon (e.g., IFNgamma), IL-2 and IL-6 cytokine receptor complexes (Kisseleva et al., 2002, gene 285:1-24; Levy et al., 2005, Nat. Rev. Mol. Cell. Biol. 3:651-662; O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). JAK1 knockout mice die perinatally due to defects in LIF receptor signaling (Kisseleva et al., 2002, gene 285:1-24; O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). Characterization of tissues derived from JAK1 knockout mice demonstrated critical roles for this kinase in the IFN, IL-10, IL-2/IL-4, and IL-6 pathways. A humanized monoclonal antibody targeting the IL-6 pathway (Tocilizumab) was recently approved by the European Commission for the treatment of moderate-to-severe rheumatoid arthritis (Scheinecker et al., 2009, Nat. Rev. Drug Discov. 8:273-274).
TYK2 is a potential target for immuno-inflammatory diseases, being validated by human genetics and mouse knock-out studies (Levy D. and Loomis C. (2007)).
TYK2 associates with the type I interferon (e.g., IFNalpha), IL-6, IL-10, IL-12 and IL-23 cytokine receptor complexes (Kisseleva et al., 2002, gene 285:1-24; Watford, W. T. & O'Shea, J. J., 2006, Immunity 25:695-697). Consistent with this, primary cells derived from a TYK2 deficient human are defective in type I interferon, IL-6, IL-10, IL-12 and IL-23 signaling. A fully human monoclonal antibody targeting the shared p40 subunit of the IL-12 and 11-23 cytokines (Ustekinumab) was recently approved by the European Commission for the treatment of moderate-to-severe plaque psoriasis (Krueger et al., 2007, N. Engl. J. Med. 356:580-92; Reich et al., 2009, Nat. Rev. Drug Discov. 8:355-356). In addition, an antibody targeting the IL-12 and IL-23 pathways underwent clinical trials for treating Crohn's Disease (Mannon et al., 2004, N. Engl. J. Med. 351:2069-79).
The role of TYK2 in the biological response to cytokines was first characterized using a mutant human cell line that was resistant to the effects of Type I interferons (IFNs) and the demonstration that IFNa responsiveness could be restored by genetic complementation of TYK2 (Velazquez et al, 1992. Cell 70, 313-322). Further in vitro studies implicated TYK2 in the signaling pathways of multiple other cytokines involved in both innate and adaptive immunity. Analysis of TYK-2 “ ” mice however revealed less profound immunological defects than were anticipated (Karaghiosoff et al, 2000. Immunity 13, 549-560; Shimoda et al, 2000. Immunity 13, 561-671). Surprisingly, TYK2 deficient mice display merely reduced responsiveness to IFNα/β and signal normally to interleukin 6 (IL-6) and interleukin 10 (IL-10), both of which activate TYK2 in vitro. In contrast, TYK2 was shown to be essential for IL-12 signaling with the absence of TYK2 resulting in defective STAT4 activation and the failure of T cells from these mice to differentiate into IFNy-producing Thl cells. Consistent with the involvement of TYK2 in mediating the biological effects of Type I IFNs and IL-12, TYK2−/− mice were more susceptible to viral and bacterial infections.
US 20100105661, WO 2007077949, WO 2007007919, WO 199965909, WO 200142246, WO 200200661, WO 2005060972 discloses JAK3 inhibitors. US 20030078277, WO 2005009389, WO 2005105788, WO2011068881, EP2420502, discloses tricyclic derivatives where as WO2011068881, EP2420502, WO0142246, WO03068157, WO9965908, WO2004047843, WO2004058749, WO2004099204, WO2004099205, WO2005037843, WO200505393, WO2005095400, WO2006096270, WO2007007919, WO2007070514, WO2007084557, WO2007117494, WO2007140222, WO2009054941, WO2009071701, WO2009155156, WO2010039939, WO2010051781,WO2010085684, WO2011003418, WO201103155 discloses bicyclic derivatives.
In aggregate, because of its restricted distribution and function within the hematopoietic cells, JAK3 has been viewed as an attractive therapeutic target for the development novel class of immunosuppressive drugs. JAK3 inhibitors would be useful in treating many autoimmune and inflammatory diseases such as, but not limited to rheumatoid arthritis, psoriasis, psoriatic arthritis, transplantation rejection, graft-versus-host disease, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, allergic diseases and asthma, and type 1 diabetes. Since JAK3-SCID patients do not exhibit pathology outside the immune system, in principle, a selective JAK3 inhibitor should have very limited and specific effects. Many of the currently used immunosuppressive drugs such as anti-metabolites, corticosteroids, and the inhibitors of calcineurin and mTOR target widely expressed molecules and hence are associated with adverse effects causing morbidity and mortality as the treatment is chronic. Similarly biologic anti-inflammatory agents such as TNF-alpha blockers are also associated with adverse events such as increase in the rate of serious infections, including tuberculosis and other opportunistic infections, injection site/infusion-related reactions, increased risk of lymphoma, the development of autoantibodies and a higher rate of congestive heart failure (CHF) in patients who already are known to have an increased risk of CHF. As a result, potent and selective JAK3 inhibitors are expected to have significant advantages over current regimens.